Keratinocytes are a cell type which synthesize keratin and are able to form a stratified squamous epithelium. The most common keratinocytes are epidermal cells of the skin. Others include the cells lining the mouth, esophagus or vagina.
Although certain types of mammalian cells have been serially cultivated for many years, it has only been recently that techniques for the serial cultivation of keratinocytes, including human epidermal cells, have been developed. These recent techniques are described in Green et al., U.S. Pat. No. 4,016,036.
In the Green et al. patent, it is disclosed that human epidermal cells or other keratinocytes can be grown in cultures with fibroblast cells treated to prevent their multiplication. Fibroblast cell density. is carefully controlled in these cultures to allow epidermal cell colony formation and growth. Keratinocytes also can be grown in the presence of fibroblast cell products as well as in the presence of fibroblast cells themselves. Using the Green et al. techniques disclosed in U.S. Pat. No. 4,016,036, it is possible to serially culture human epidermal cells and expand the number in the primary culture by many-fold.
In more recent work by Green, it has been discovered that agents known to increase the level of cellular cyclic-AMP can provide a dramatic effect on the growth of epithelial cells. Thus, agents such as cholera toxin, dibutyryl cyclic-AMP, methyl isobutyl xanthine and isoproterenol are employed to increase the multiplication of human epidermal cells in serial cultivation.
It is also known that disaggregated epidermal cells obtained directly from the epidermis or from briefly cultured cells can be applied to a graft bed and reconstitute an epidermis. See Billingham, R. E. and Reynolds, J., Brit. J. Plastic Surg., 5:25-36 (1952); and Yuspa, S. H., Morgan, D. L., Walker, R. J. and Bates, R. R. J. Invest. Dermatol., 55:379-389 (1970). Nevertheless, use of the disaggregated epidermal cells is probably not the most efficient method of grafting cultured cells. Since the stratification in cell cultures is such that the multiplying cells lie on the surface of the dish, it would be desirable to retain this polarity in the graft by applying an intact culture-grown epithelium, rather than disaggregated cells, of which a large fraction would be incapable of multiplication.
A method for employing a culture epithelium has been disclosed in which cultures are grown on transplantable collagen surfaces. See Worst, P. K. M., Valentine, E. A. and Fusenig, N. E., J. Natl. Cancer Institute, 53:1061-1064 (1974). Although it would be highly desirable, there has not heretofore been a method for detaching confluent epidermal sheets from the surface of a petri dish without disassociating the cells.